Multivalent immunogenic compositions against human papillomavirus

Abstract

The present invention relates to a multivalent human papillomavirus (HPV) immunogenic composition and its use for preventing HPV-associated disease or infection. The multivalent HPV immunogenic composition comprises HPV virus-like particles assembled from L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, as well as one or more HPV virus-like particles assembled from L1 proteins of other pathogenic HPV types. In one embodiment, the one or more other pathogenic HPV types are selected from HPV types 35, 39, 51, 56, and 59. In one embodiment, at least one of the HPV virus-like particles is a chimeric HPV virus-like particle, and the chimeric HPV virus-like particle comprises one or more chimeric HPV L1 proteins.

Description

[Technical Field]

[0001] The present invention relates to multivalent immunogenic compositions and uses thereof for preventing human papillomavirus (HPV)-associated disease or infection. [Background technology]

[0002] Papillomaviruses (PV) belong to the family papillomaviridae and cause papillomas in humans, cattle, dogs, and rabbits. One of its members, human papillomavirus (HPV), is a non-enveloped DNA virus. The genome of this virus is a double-stranded, closed circular DNA approximately 7.2 to 8 kb in size, with eight open reading frames, which can be divided into three regions based on their functions: (1) an approximately 4.5 kb early region (E) encoding six nonstructural proteins, E1, E2, E4 to E7, involved in viral replication, transcription, and transformation; (2) an approximately 2.5 kb late region (L) encoding the major capsid protein L1 and the minor capsid protein L2; and (3) a long control region (LCR) approximately 800 to 900 bp in length, located between the end of the L region and the beginning of the E region, which does not encode any proteins and acts as a DNA replication and expression regulatory element.

[0003] The L1 and L2 proteins are synthesized late in the HPV infection cycle. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is the minor capsid protein. 72 L1 protein pentamers form the outer shell of the icosahedral HPV particle (45-55 nm in diameter), which encapsulates the closed circular double-stranded DNA. The L2 protein is located inside the L1 protein (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, X.S., R.L. Garcea, Mol. Cell. 5(3):557-567, 2000).

[0004] The ORF of the L1 protein, the most conserved gene in the PV genome, can be used to identify new PV types. A new PV type is identified when its entire genome is cloned and its L1 ORF DNA sequence differs by more than 10% from the closest known PV type. Homology with a difference of 2% to 10% is defined as a different subtype, and differences of less than 2% are defined as different variants of the same subtype (E.-M. de Villiers et al., Virology 324 (2004) pp. 17-27).

[0005] During the late stages of HPV infection, newly synthesized L1 protein in the cytoplasm is transported to the nucleus of terminally differentiated keratinocytes and, together with L2 protein, packages replicated HPV genomic DNA to form infectious virus (Nelson, LM et al., 2002, Nuclear import strategies of high-risk HPV16 L1 major capsid protein. J. Biol. Chem. 277:23958-23964). This suggests that nuclear import of L1 protein plays a crucial role in HPV infection and development. The ability of the virus to enter the nucleus is determined by the nuclear localization signal (NLS) at the C-terminus of the HPV L1 protein, which is characterized by a high concentration of basic amino acids (Garcia-Bustos, J. et al., 1991, Nuclear protein localization. Biochimica et Biophysica Acta 1071:83-101).

[0006] Fifteen high-risk (HR) HPV types can cause cancer of the cervix, anus, penis, vagina, vulva, and oropharynx, with HPV-16 and HPV-18 being the most common causes of cancer, accounting for approximately 70% of cervical cancers, and other HR-HPV types (types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) causing the rest. HPV-16 accounts for approximately 95% of HPV-positive oropharyngeal cancers (OPC). The persistent low-risk genotypes HPV-6 and HPV-11 cause most anogenital warts and respiratory papillomas but are largely unrelated to cancer (Human Papillomavirus in Cervical Cancer and Oropharyngeal Cancer: One Cause, Two Diseases Tara A. Berman and John T. Schiller, PhD2 Cancer 2017;123:2219-29).

[0007] The L1 protein can be recombinantly expressed in poxvirus, baculovirus, or yeast systems and then self-assembles to form virus-like particles (VLPs) containing approximately 72 L1 proteins similar to viral capsids. VLPs are symptomless. VLPs induce neutralizing antibodies in inoculated animals and protect experimental animals from subsequent challenge with infectious virus. Therefore, VLPs appear to be excellent candidates for papillomavirus vaccines (Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, XS, RL Garcea, Mol. Cell. 5(3):557-567, 2000).

[0008] Glaxo's CERVARIX®, a bivalent recombinant HPV vaccine, contains recombinant HPV type 16 L1 protein and recombinant HPV type 18 L1 protein. The L1 protein is obtained by expressing a recombinant baculovirus expression vector system in insect cells of a nocturnal moth (Trichoplusia ni). The L1 protein self-assembles into virus-like particles for the prevention of in situ cervical cancer, grade 2 or 3 cervical intraepithelial neoplasia, and adenocarcinoma caused by HPV types 16 and 18, and grade 1 cervical intraepithelial neoplasia (oncogenic) in women aged 9 to 25 years (https: / / www.fda.gov / downloads / BiologicsBloodVaccines / Vaccines / ApprovedProducts / UCM186981.pdf).

[0009] GARDASIL® is a quadrivalent recombinant human papillomavirus (types 6, 11, 16, and 18) vaccine marketed by Merck for the prevention of cervical cancer, genital warts, and precancerous or abnormal growths caused by HPV types 6, 11, 16, and 18 in girls and women ages 9 to 26, and for the prevention of anal cancer, genital warts, and precancerous or abnormal growths caused by HPV types 6, 11, 16, and 18 in boys and men ages 9 to 26 (https: / / www.fda.gov / vaccines-blood-biologics / vaccines / gardasil).

[0010] GARDASIL® 9 is a nonavalent recombinant human papillomavirus vaccine marketed by Merck containing virus-like particles of the L1 protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, where the L1 protein is produced by fermentation of Saccharomyces cerevisiae and self-assembly into VLPs. This is to prevent cervical, vulvar, vaginal, and anal cancer caused by HPV types 16, 18, 31, 33, 45, 52, and 58 in girls and women aged 9-45, genital warts (condyloma acuminata) caused by HPV types 6 and 11, and precancerous or growth abnormalities caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and to prevent HPV types 16, 18, 31, 33, 45, 52, and 58 in boys and men aged 9-45. It is used to prevent anal cancer caused by HPV types 8, 31, 33, 45, 52, and 58, genital warts (condyloma acuminata) caused by HPV types 6 and 11, and precancerous or developmentally abnormal lesions caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 (https: / / www.fda.gov / vaccines-blood-biologics / vaccines / gardasil-9).

[0011] The GARDASIL® 9 instructions disclose that approximately 70% of cervical cancers are caused by HPV types 16 and 18, with the remaining 20% ​​of cases attributed to types 31, 33, 45, 52, and 58; therefore, GARDASIL® 9 prevents 90% of cervical cancers (https: / / www.fda.gov / BiologicsBloodVaccines / Vaccines / ApprovedProducts / ucm426445.htm).

[0012] Industrial production of virus-like particles is important for HPV vaccine development. Common systems for producing virus-like particles are mainly classified into eukaryotic and prokaryotic expression systems.

[0013] Commonly used eukaryotic expression systems include poxvirus expression systems, insect baculovirus expression systems, and yeast expression systems. HPV L1 protein expressed in eukaryotic expression systems can spontaneously assemble into virus-like particles because its native conformation is not significantly disrupted, but the yield is low. Although the yield of HPV L1 protein expressed in prokaryotic expression systems, mainly E. coli expression systems, is high, it is mainly in the form of inclusion bodies, and this form of protein cannot be easily purified, thus complicating the production process.

[0014] Therefore, there remains a need to obtain high yields of HPV virus-like particles in order to obtain an HPV multivalent vaccine that provides broad protection against HPV-associated diseases or infections, including those caused by HPV types not currently covered by commercially available vaccines. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Patent No. 6,361,778 B1 [Non-patent literature]

[0016] [Non-licensed Document 1] Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, XS, RLGarcea, Mol.Cell.5(3): pages 557~567, 2000 [Non-licensed Document 2] E.-M. de Villiersら / Virology 324 (2004) pages 17~27 [Non-licensed Document 3] Nelson, LMら, 2002, nuclear import strategies of high risk HPV16 L1 major capsid protein. J. Biol. Chem. 277:23958~23964 pages [Non-licensed Document 4] Garcia-Bustos, J.ら, 1991, Nuclear protein localization. Biochimica et Biophysica Acta 1071:83~101 pages [Non-licensed Document 5] Human Papillomavirus in Cervical Cancer and Oropharyngeal Cancer: One Cause, Two Diseases Tara A. Bermanand John T. Schiller, PhD2 Cancer 2017;123:2219~29 pages [Non-licensed Document 6] Structure of Small Virus-like Particles Assembled from the L1 Protein of Human Papillomavirus 16 Chen, XS, RL Garcea, Mol. Cell. 5(3): pp. 557~567, 2000 [Non-licensed Document 7] https: / / www.fda.gov / downloads / BiologicsBloodVaccines / Vaccines / ApprovedProducts / UCM186981.pdf [Non-licensed document 8] https: / / www.fda.gov / vaccines-blood-biologics / vaccines / gardasil

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0017] In one aspect, the invention provides a multivalent HPV immunogenic composition for preventing HPV-associated disease or infection, comprising HPV virus-like particles assembled from L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, as well as one or more HPV virus-like particles assembled from L1 proteins of other pathogenic HPV types.

[0018] In another aspect, the invention provides a method for preventing an HPV-associated disease or infection comprising administering to a subject a multivalent HPV immunogenic composition.

[0019] In another aspect, the present invention provides the use of a multivalent HPV immunogenic composition in the preparation of a vaccine or medicament for the prevention of HPV-associated disease or infection. [Brief explanation of the drawings]

[0020] [Figure 1A] Figure 1 shows the expression of L1 protein of HPV6 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1B] Figure 1 shows the expression of L1 protein of HPV11 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1C] Figure 1 shows the expression of L1 protein of HPV16 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1D] Figure 1 shows the expression of L1 protein of HPV18 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1E] Figure 1 shows the expression of L1 protein of HPV31 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1F] Figure 1 shows the expression of L1 protein of HPV33 L1. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1G] Figure 1 shows the expression of L1 protein of HPV35 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1H] Figure 1 shows the expression of L1 protein of HPV39 L1:59C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1I] Figure 1 shows the expression of L1 protein of HPV45 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1J] Figure 1 shows the expression of L1 protein of HPV51 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1K] Figure 1 shows the expression of L1 protein of HPV52 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1L] Figure 1 shows the expression of L1 protein of HPV56 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1M] Figure 1 shows the expression of L1 protein of HPV58 L1:33C. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 1N] Figure 1 shows the expression of L1 protein of HPV59 L1. M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. [Figure 2A] FIG. 1 shows a transmission electron microscope image of HPV6 L1:33C virus-like particles. [Figure 2B] FIG. 1 shows a transmission electron microscope image of HPV11 L1:33C virus-like particles. [Figure 2C] FIG. 1 shows a transmission electron microscope image of HPV16 L1:33C virus-like particles. [Figure 2D] FIG. 1 shows a transmission electron microscope image of HPV18 L1:33C virus-like particles. [Figure 2E] FIG. 1 shows a transmission electron microscope image of HPV31 L1:33C virus-like particles. [Figure 2F] FIG. 1 shows a transmission electron microscope image of HPV33 L1 virus-like particles. [Figure 2G] FIG. 1 shows a transmission electron microscope image of HPV35 L1:33C virus-like particles. [Figure 2H] FIG. 1 shows a transmission electron microscope image of HPV39 L1:59C virus-like particles. [Figure 2I]FIG. 1 shows a transmission electron microscope image of HPV45 L1:33C virus-like particles. [Figure 2J] FIG. 1 shows a transmission electron microscope image of HPV51 L1:33C virus-like particles. [Figure 2K] FIG. 1 shows a transmission electron microscope image of HPV52 L1:33C virus-like particles. [Figure 2L] FIG. 1 shows a transmission electron microscope image of HPV56 L1:33C virus-like particles. [Figure 2M] FIG. 1 shows a transmission electron microscope image of HPV58 L1:33C virus-like particles. [Figure 2N] FIG. 1 shows a transmission electron microscope image of HPV59 L1 virus-like particles. [Figure 3] Figure 1 shows pseudovirus neutralization titers in mice immunized with a composition of virus-like particles of 14 HPV types. Number of mice N=10. GMT: geometric mean titer. [Figure 4] Figure 1 shows the expression of C-terminally truncated HPV16L1 (1-474). M: marker; L: cell lysate; ES: supernatant collected after centrifugation of the lysate. DETAILED DESCRIPTION OF THE INVENTION

[0021] In one aspect, the present invention provides a multivalent immunogenic composition for preventing papillomavirus-associated disease or infection. In one embodiment, the papillomavirus can be human papillomavirus. In another embodiment, the papillomavirus can be canine papillomavirus or rabbit papillomavirus.

[0022] In one aspect, the invention provides a multivalent HPV immunogenic composition for preventing HPV-associated disease or infection, comprising HPV virus-like particles assembled from L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and one or more HPV virus-like particles assembled from L1 proteins of other pathogenic HPV types.

[0023] In one embodiment, the L1 protein of each HPV type can be a native L1 protein, a non-native L1 protein, or a chimeric HPV L1 protein. In one embodiment, the HPV virus-like particles can be assembled from a single type of HPV L1 protein to form a monovalent HPV virus-like particle, or can be assembled from multiple types of HPV L1 proteins to form a multivalent HPV virus-like particle.

[0024] In one embodiment, the one or more other pathogenic HPV types are selected from HPV types 35, 39, 51, 56, and 59.

[0025] In one embodiment, at least one of the HPV virus-like particles is a chimeric HPV virus-like particle, wherein the chimeric HPV virus-like particle comprises one or more chimeric HPV L1 proteins, wherein the chimeric HPV L1 proteins comprise, from its N-terminus to its C-terminus: a. an N-terminal fragment derived from an L1 protein of a first papillomavirus type, wherein the L1 protein of the first papillomavirus type is selected from HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, or 58, and wherein the N-terminal fragment maintains the immunogenicity of the L1 protein of the corresponding HPV type; b. a C-terminal fragment derived from an L1 protein of a second papillomavirus type, wherein the L1 protein of the second papillomavirus type has a better expression level and solubility compared to the L1 proteins of other types; Including, The chimeric L1 protein of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, or 58 has the immunogenicity of the L1 protein of the corresponding HPV type.

[0026] In one embodiment, the chimeric HPV virus-like particles can be assembled from a single type of chimeric HPV L1 protein to form a monovalent HPV virus-like particle, or can be assembled from multiple types of chimeric HPV L1 proteins to form a multivalent HPV virus-like particle.

[0027] In one embodiment, the C-terminal fragment and the N-terminal fragment can be freely combined as needed. In one embodiment, the chimeric HPV L1 protein can include one or more C-terminal fragments. The multiple C-terminal fragments can be the same or different.

[0028] In one embodiment, the N-terminal fragment is a fragment obtained by truncating the C-terminus of the native sequence of the L1 protein of a first papillomavirus type at any amino acid position within its α5 region, and a fragment having at least 98% identity thereto, and the C-terminal fragment is a fragment obtained by truncating the N-terminus of the native sequence of the L1 protein of a second papillomavirus type at any amino acid position within its α5 region, and a functional variant obtained by further mutation, deletion, and / or addition to the fragment.

[0029] In another embodiment, the N-terminal fragment has at least 98.5%, 99%, 99.5%, or 100% identity to a fragment obtained by truncating the C-terminus of the native sequence of the L1 protein of the first papillomavirus type at any amino acid position within its α5 region.

[0030] In one embodiment, the C-terminal fragment comprises one or more nuclear localization sequences.

[0031] In one embodiment, the L1 protein of the second papillomavirus type is selected from the L1 protein of HPV type 1, 2, 3, 4, 6, 7, 10, 11, 13, 16, 18, 22, 26, 28, 31, 32, 33, 35, 39, 42, 44, 45, 51, 52, 53, 56, 58, 59, 60, 63, 66, 68, 73, or 82.

[0032] Preferably, said L1 protein of a second papillomavirus type is selected from the L1 protein of HPV types 16, 28, 33, 59, or 68.

[0033] More preferably, said L1 protein of the second papillomavirus type is selected from the L1 protein of HPV type 33 or HPV type 59.

[0034] In one embodiment, the L1 protein of the second papillomavirus type is an HPV33 L1 protein, and the C-terminal fragment is SEQ ID NO: 2, or a fragment having a length of m1 amino acids, preferably a fragment covering amino acids at positions 1 to m1 of SEQ ID NO: 2 (where m1 is an integer of 8 to 26), or the C-terminal fragment is SEQ ID NO: 135, or a fragment having a length of m2 amino acids, preferably a fragment covering amino acids at positions 1 to m2 of SEQ ID NO: 135 (where m2 is an integer of 13 to 31).

[0035] In one embodiment, the C-terminal fragment of HPV33 L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV33 L1 protein has two nuclear localization sequences. In one embodiment, the amino acid sequence having amino acid numbers 7 to 8 (KR) and the amino acid sequence having amino acid numbers 20 to 23 (KRKK) of SEQ ID NO: 2 are the nuclear localization sequences of the C-terminal fragment of HPV33 L1 protein.

[0036] In one embodiment, the L1 protein of the second papillomavirus type is HPV59 L1 protein, and the C-terminal fragment is SEQ ID NO: 13 or a fragment having a length of n amino acids, preferably a fragment covering amino acids 1 to n (n is an integer from 16 to 38) of SEQ ID NO: 13.

[0037] In one embodiment, the C-terminal fragment of HPV59 L1 protein has a nuclear localization sequence. In another embodiment, the C-terminal fragment of HPV59 L1 protein has two nuclear localization sequences. In some embodiments, the chimeric HPV L1 protein comprises one or more C-terminal fragments of HPV59 L1 protein. The multiple C-terminal fragments of HPV59 L1 protein may be the same or different. In one embodiment, the amino acid sequence (RKR) at positions 14 to 16 of SEQ ID NO: 13 and the amino acid sequence (KRVKRRK) at positions 28 to 34 of SEQ ID NO: 13 are nuclear localization sequences of the C-terminal fragment of HPV59 L1 protein.

[0038] In one embodiment, the chimeric HPV L1 protein comprises both a C-terminal fragment of an HPV33 L1 protein and a C-terminal fragment of an HPV59 L1 protein.

[0039] In one embodiment, the N-terminal fragment of the HPV6 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 1 at any amino acid position within the alpha 5 region.

[0040] The N-terminal fragment of the HPV11 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID NO: 14 at any amino acid site within the alpha 5 region.

[0041] The N-terminal fragment of the HPV16 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID NO: 27 at any amino acid site within the alpha 5 region.

[0042] The N-terminal fragment of the HPV18 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 40 at any amino acid site within the alpha 5 region.

[0043] The N-terminal fragment of the HPV31 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID NO: 53 at any amino acid site within the alpha 5 region.

[0044] The N-terminal fragment of the HPV35 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 69 at any amino acid site within the alpha 5 region.

[0045] The N-terminal fragment of the HPV39 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence shown in SEQ ID NO: 82 at any amino acid site within the alpha 5 region.

[0046] The N-terminal fragment of the HPV45 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 95 at any amino acid site within the alpha 5 region.

[0047] The N-terminal fragment of the HPV51 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 108 at any amino acid site within the alpha 5 region.

[0048] The N-terminal fragment of the HPV52 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 121 at any amino acid site within the alpha 5 region.

[0049] The N-terminal fragment of the HPV56 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity with a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 134 at any amino acid site within the alpha 5 region.

[0050] The N-terminal fragment of the HPV58 L1 protein has 98%, 98.5%, 99%, 99.5%, 99%, or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 147 at any amino acid site within the alpha 5 region.

[0051] In one embodiment, the C-terminus of the N-terminal fragment is connected to the N-terminus of the C-terminal fragment directly or via a linker.

[0052] The linker does not affect the immunogenicity of the N-terminal fragment and does not affect the expression level or solubility of the protein. In one embodiment, the N-terminal fragment and the C-terminal fragment are connected via a linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the linker is an artificial sequence. In another embodiment, the linker is a naturally occurring sequence in the HPV L1 protein. In another embodiment, the linker may be a partial sequence of the HPV33 L1 protein. In another embodiment, the linker may be a partial sequence of the HPV59 L1 protein.

[0053] In one embodiment, when the C-terminus of the N-terminal fragment is joined to the N-terminus of the C-terminal fragment, the contiguous amino acid sequence RKFL is present within ±4 amino acid positions from the joining point, and preferably the contiguous amino acid sequence LGRKFL is present within ±6 amino acid positions from the joining point.

[0054] In some embodiments, the chimeric HPV L1 proteins of chimeric HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58 have 98%, 98.5%, 99%, 99.5%, or 100% identity to SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:29, SEQ ID NO:42, SEQ ID NO:55, SEQ ID NO:71, SEQ ID NO:84, SEQ ID NO:97, SEQ ID NO:110, SEQ ID NO:123, SEQ ID NO:136, and SEQ ID NO:149, respectively, and the HPV type 33 L1 proteins and HPV type 59 L1 proteins have 98%, 98.5%, 99%, 99.5%, or 100% identity to SEQ ID NO:66 and SEQ ID NO:160, respectively.

[0055] In one embodiment, at least one of said HPV virus-like particles is composed of a single type of chimeric HPV L1 protein, preferably a single type of chimeric HPV L1 protein having the same amino acid sequence.

[0056] In one embodiment, the chimeric HPV virus-like particle is an icosahedron containing 72 pentamers of the chimeric HPV L1 protein. In one embodiment, the HPV virus-like particle has correctly formed disulfide bonds and therefore has a good native conformation. In one embodiment, the chimeric HPV virus-like particle self-assembles in an in vivo expression system.

[0057] In one embodiment, the multivalent HPV immunogenic composition further comprises a physiologically acceptable carrier and, optionally, an adjuvant, hi one embodiment, the adjuvant comprises one or more of an aluminum salt, a lipid A derivative, and an ISCOM.

[0058] In one embodiment, the adjuvant is an aluminum phosphate adjuvant.

[0059] In one aspect, the present invention provides a method for preventing HPV-associated disease or infection, comprising administering a multivalent HPV immunogenic composition to a subject. The prevention can be considered a treatment, although the terms can be used interchangeably. In one embodiment, the subject is a human.

[0060] In one aspect, the present invention provides the use of the above multivalent HPV immunogenic composition in the preparation of a vaccine or medicament for preventing HPV-related disease or infection.

[0061] Papillomavirus L1 protein expressed in eukaryotic expression systems can spontaneously assemble into virus-like particles, but the low expression levels make it unsuitable for large-scale production.

[0062] The sequence of the L1 protein for each HPV type can be easily obtained from https: / / www.uniprot.org. For a given HPV type, the L1 protein may be derived from different lineages and therefore may have multiple versions of its amino acid sequence, but any version of the natural sequence can be used in the present invention. The sequence of the HPV L1 protein for a given type used during the conception and design of the present invention may differ from the sequence used in the examples below, but such differences do not affect the inventors' decisions and conclusions.

[0063] It is generally recognized by those skilled in the art that the C-terminus of the L1 protein does not contain major neutralizing antigen epitopes. Therefore, attempts have been made to increase expression by truncating the C-terminus of the HPV L1 protein. For example, U.S. Patent No. 6,361,778 B1 (Glaxo) discloses that truncating 1-34 amino acids, preferably 26 amino acids, from the C-terminus of the HPV16 L1 protein increases VLP yield several-fold, preferably at least 10-fold, and particularly approximately 10-100-fold. Inspired by this, the present inventors attempted to truncate 31 amino acids from the C-terminus of the HPV16 L1 protein and designated this truncated protein HPV16 L1(1-474). While the protein exhibits high expression levels, it is poorly soluble and difficult to extract and purify (see Comparative Example).

[0064] The poor solubility of the protein due to this truncation may be caused by the deletion of the nuclear localization sequence located at the C-terminus, but the present invention is not bound by this speculation. During research and production, the present inventors discovered that the HPV16 L1 protein, HPV28 L1 protein, HPV33 L1 protein, HPV59 L1 protein, and HPV68 L1 protein have better expression levels and solubility than other HPV L1 proteins. Encouraged by this discovery, the present inventors replaced the C-terminus of a specific HPV L1 protein that is less extractable or has lower solubility with the C-terminus of a specific HPV L1 protein that has better expression levels and solubility. That is, the present inventors have constructed chimeric proteins comprising, from the N-terminus to the C-terminus, an N-terminal fragment derived from an L1 protein of a first papillomavirus type (e.g., HPV L1 protein) that provides the immunogenicity of the first papillomavirus type (e.g., HPV), and a C-terminal fragment derived from an L1 protein of a second papillomavirus type (e.g., HPV L1 protein) that provides better expression levels and solubility characteristics. These two fragments can be connected directly or via a linker.

[0065] The length of the N-terminal fragment of HPV L1 protein that maintains the immunogenicity of the first type of L1 protein and is suitable for ensuring VLP formation is determined. The following report relates to epitope research on common HPV types.

[0066] Sunanda Baidya et al. reported that epitopes from L1 proteins 48EEYDLQFIFQLCKITLTA65, 45RHGEEYDLQFIFQLCKITLTA65, 63LPDPNKF69, 79PETQRLVWAC88, 36PVPGQYDA43, 77YNPETQRLVWAC88, 188DTGYGAMD195, 36PVPGQYDATK45, 45KQDIPKVSAYQYRVFRV61, 130RDNVSVDYKQTQLCI144, and 49YSRHVEEYDLQFIF62 can be used as tools for designing HPV16 and 18 vaccines (see, “Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18,” Bioinformation 13(3):86-93, March 2017, which is incorporated herein by reference in its entirety).

[0067] Katharina Slupetzky et al. reported that regions located near aa 282-286 and 351-355 of HPV-16 contribute to neutralizing epitopes, with the latter being the immunodominant site (see Chimeric papilloma virus-like particles expressing a foreign epitope on capsid surface loops, Journal of General Virology (2001) 82, pp. 2799-2804, which is incorporated herein by reference in its entirety).

[0068] Brooke Bishop et al. prepared three variants of HPV11, 16, 18, and 35 L1 proteins: one with a deletion of 9 amino acids at its N-terminus, one with a deletion of α4 (corresponding to amino acid residues 404-436 of HPV16), and one with a deletion of 31 amino acids at its C-terminus, and reported that the former two did not assemble into VLPs, but this phenomenon was not reported for the latter one (Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins Understanding the Specificity of Neutralising Monoclonal Antibodies, The Journal of Biological Chemistry, 282, pp. 31803-31811, which is incorporated herein by reference in its entirety). The α-helix, β-fold sheet, and loop regions of each type of HPV L1 protein can be conveniently determined using sequence analysis software commonly used in the art. In that case, the α-helical region contains an α1 region, an α2 region, an α3 region, an α4 region, and an α5 region.

[0069] [ka]

[0070] The present inventors performed sequence alignment of the L1 proteins of 14 HPV types (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) and then performed secondary structure prediction according to the above-cited literature (Crystal Structures of Four Types of Human Papillomavirus L1 Capsid Proteins Understanding the Specificity of Neutralizing Monoclonal Antibodies, The Journal of Biological Chemistry, 282, pp. 31803-31811). The results are shown below, and the portions between the downward arrows correspond to the regions deleted to prepare the variants in the literature.

[0071] [ka] [ka]

[0072] In addition to the method used by the inventors for sequence alignment, protein secondary structure prediction software that can be used for prediction includes, but is not limited to: 1. JPred:http: / / www.compbio.dundee.ac.uk / jpred / index.html 2. ProtPredict:http: / / predictprotein.org 3. PsiPred: http: / / bioinf.cs.ucl.ac.uk / psipred 4. SCRATCH-1D:http: / / download.igb.uci.edu 5. Nnpredict:http: / / www.cmpharm.ucsf.edu / ~nomi / nnpredict 6. predictprotein:http: / / www.embl-heidelberg.de / predictprotein / SOPMA http: / / www.ibcp.fr / predict.html 7. SSPRED:http: / / www.embl-heidelberg.de / sspred / ssprd_info.html.

[0073] In one embodiment of the present invention, the inventors determine the length of an N-terminal fragment derived from the L1 protein of a first HPV type as follows: the native sequence of the L1 protein is truncated within its α5 region and its adjacent region, and the sequence from its N-terminus to the newly generated C-terminus within the α5 region is retained. Such a truncated sequence ensures that it has the immunogenicity of the first type and the ability to form VLPs.

[0074] The N-terminal fragment of the HPV L1 protein derived from the first type can be further modified as long as it retains the immunogenicity of the first type and has the ability to form VLPs.

[0075] The length of the C-terminal fragment of the HPV L1 protein from the second type is determined as follows: the native sequence of the L1 protein is truncated within its α5 region and its adjacent regions, and then the newly generated N- to C-terminal sequence within the α5 region is retained. Such a truncated sequence does not contain major neutralizing antigenic epitopes and therefore does not interfere with the immunogenicity of the resulting chimeric protein.

[0076] The C-terminal fragment of the HPV L1 protein from the second type can be further mutated, deleted, and / or added, preferably while retaining at least one of its nuclear localization sequences. Yang et al. predicted the nuclear localization sequences of 107 HPV subtypes (Yang et al., "Predicting the nuclear localization signals of 107 types of HPV L1 proteins by bioinformatic analysis." Geno. Prot. Bioinfo. Vol. 4 No. 1, 2006, incorporated herein by reference in its entirety). The nuclear localization sequence of each type of HPV L1 protein can be conveniently determined using sequence analysis software commonly used in the art.

[0077] The connection between the N-terminal fragment and the C-terminal fragment occurs at the newly generated C-terminus of the former and the newly generated N-terminus of the latter. This connection may be direct or via a linker. The site where the connection occurs is defined as the origin coordinate, with the N-terminal side of the origin considered negative, while the C-terminal side is considered positive.

[0078] The sequence of amino acids 453 to 469 of the HPV6 L1 protein and the corresponding sequences of L1 proteins of other HPV types are shown below. These sequences overlap with each other in the α5 region. It can be seen that these sequences are very similar. The number in parentheses represents the position of the last amino acid residue in the listed sequence. In the case of HPV45, an additional 26 amino acids are present at the N-terminus of the L1 protein of some HPV45 strains, while the additional 26 amino acids are not present at the N-terminus of other HPV45 strains, and therefore the number is shown as (478)+26.

[0079] HPV6 ELDQYPLGRKFLLQSGY(469)

[0080] HPV11 ELDQFPLGRKFLLQSGY(470)

[0081] HPV16 DLDQFPLGRKFLLQAGL(474)

[0082] HPV18 DLDQYPLGRKFLVQAGL(475)

[0083] HPV31 DLDQFPLGRKFLLQAGY(475)

[0084] HPV35 DLDQFPLGRKFLLQAGL(472)

[0085] HPV39 ELDQFPLGRKFLLQARV(474)

[0086] HPV45 DLDQYPLGRKFLVQAGL(478)+26

[0087] HPV51 DLDQFALGRKFLLQVGV(474)

[0088] HPV52 DLDQFPLGRKFLLQAGL(478)

[0089] HPV56 DLDQFPLGRKFLMQLGTRS(474)

[0090] HPV58 DLDQFPLGRKFLLQSGL(473)

[0091] HPV33 DLDQFPLGRKFLLQAGL(473)KAKPKLKRAAPTSTRTSSAKRKKVKK In the formula, KR at positions 480 to 481 and KRKK at positions 493 to 496 are nuclear localization sequences.

[0092] HPV59 DLDQFPLGRKFLLQLGA(475)RPKPTIGPRKRAAPAPTSTPSPKRVKRRKSSRK In the formula, RKR at positions 484 to 486 and KRVKRRK at positions 498 to 504 are nuclear localization sequences.

[0093] In one embodiment of the present invention, the inventors took advantage of the sequence similarity between HPV types in the α5 region and adjacent regions to complete C-terminal substitution of the L1 protein between different HPV types.

[0094] In the most preferred embodiment of the present invention, the inventors have pointed out that the L1 proteins of each HPV type have the tetrapeptide RKFL, or more preferably the hexapeptide LGRKFL, at a similar position. The inventors have cleverly utilized this highly conserved sequence to position the chimeric protein attachment point at any amino acid position within this oligopeptide. On the one hand, the sequence starting from the N-terminus of the chimeric protein to RKFL or LGRKFL is identical to the sequence of the N-terminal fragment derived from the L1 protein of the first type, while on the other hand, the sequence starting from RKFL or LGRKFL to the C-terminus of the chimeric protein is identical to the sequence of the C-terminal fragment derived from the L1 protein of the second type.

[0095] The chimeric proteins so produced maintain a high degree of similarity to the natural HPV L1 protein and can be expected to perform well in manufacturing and even in subsequent medical or prophylactic procedures.

[0096] Those skilled in the art will appreciate that different strains with different native sequences of a given HPV type exist, and chimeric proteins constructed using different strains are also within the scope of the present invention.

[0097] Due to the high similarity between L1 proteins of different HPV types, those skilled in the art will understand that even if, during the construction of a chimeric protein material, an N-terminal fragment derived from an L1 protein of a first type is extended by more amino acid residues toward the C-terminus, or a C-terminal fragment derived from an L1 protein of a second HPV virus type is extended by more amino acid residues toward the N-terminus, it will still be possible to form a chimeric protein structurally identical to that of the present invention, since the amino acids at the corresponding positions will be identical or similar. Chimeric proteins thus formed will still fall within the scope of the present invention.

[0098] Based on the chimeric protein of the above embodiment, those skilled in the art can understand that variants of the chimeric protein can be formed by mutation, deletion, and / or addition of amino acid residues. These variants may have the immunogenicity of the first type of L1 protein, can form VLPs, and have good yield and solubility. Chimeric proteins formed in this way are also included within the scope of the present invention.

[0099] Beneficial Effects of the Invention Commonly used expression systems for producing virus-like particles are classified into eukaryotic and prokaryotic expression systems. Papillomavirus L proteins expressed in eukaryotic expression systems can spontaneously assemble into virus-like particles, but have the disadvantage of low expression levels and are therefore not suitable for mass production. Papillomavirus L proteins expressed in prokaryotic expression systems often lose their native conformation, requiring in vitro processing to obtain virus-like particles and resulting in low yields, making them difficult to use industrially.

[0100] The present invention relates to a method for modifying the C-terminus of the L protein of a papillomavirus (e.g., human papillomavirus) by, for example, substituting it with the C-terminal fragment of the HPV16 L1 protein, the HPV28 L1 protein, the HPV33 L1 protein, the HPV59 L1 protein, or the HPV68 L1 protein, and thus the method can be used in an expression system (e.g., a host cell, e.g., an insect cell) to improve the expression level and solubility of the papillomavirus L protein in the expression system (e.g., a host cell, e.g., an insect cell), which can be used for mass production of vaccines, such as HPV vaccines.

[0101] The present inventors have found that the expression levels and solubility of the HPV16, HPV28, HPV33, HPV59, and HPV68 L1 proteins are increased compared to the L1 proteins of other HPV types, and that the increased expression levels and solubility of these proteins depend on the C-terminal sequence of the HPV L1 protein. Most of the 107 HPV L1 proteins have a nuclear localization sequence at their C-terminus, and the C-terminal sequences share some similarity.

[0102] For papillomavirus L proteins that are currently unexpressible, expressed at very low levels, or insoluble after expression, replacing the C-terminal fragment with that of HPV16 L1 protein, HPV28 L1 protein, HPV33 L1 protein, HPV59 L1 protein, or HPV68 L1 protein enables soluble expression and subsequent purification. This strategy can be used for the mass production of multivalent vaccines (e.g., HPV vaccines) and can provide more thorough protection against a wide range of papillomavirus infections, especially HPV.

[0103] For mass production purposes, the expression level and solubility of HPV L1 protein in insect cells must be increased. In addition, virus-like particles assembled from HPV L protein lack good conformation in yeast cells due to a failure to form proper disulfide bonds.

[0104] In the case of HPV L1 protein, which is poorly expressed and insoluble in insect cells, modifying its C-terminal fragment to the C-terminal fragment of HPV type 33 or 59 L1 protein can then significantly increase the expression level and solubility, and therefore it can be used for mass production of HPV vaccines.

[0105] HPV L1 proteins, such as HPV16, HPV28, and HPV68 L1 proteins, have better expression and solubility in insect cells than other HPV L1 proteins, but their expression level and solubility need to be further improved to achieve mass production of vaccines. In the present invention, for example, after modifying the C-terminal fragment of HPV16 L1 protein with the C-terminal fragment of HPV33 L1 protein, the expression level and solubility of the modified chimeric HPV16 protein are improved, which is useful for mass production of HPV vaccines.

[0106] In summary, the chimeric HPV L1 protein showed significantly higher expression levels and solubility in insect cells compared with the unmodified HPV L1 protein, which can be used for the mass production of HPV vaccines. In addition, the chimeric HPV L1 protein can correctly form disulfide bonds and therefore can assemble into HPV virus-like particles with a good three-dimensional structure in insect cells. This can improve the immunogenicity of HPV virus-like particles and induce a better immune response.

[0107] The multivalent vaccine of the present invention can be used to prevent diseases or infections associated with a wide range of HPVs, including those HPV types that are not currently preventable.

[0108] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. To facilitate the understanding of the present invention, the following terms are referred to below in their ordinary meanings.

[0109] As used in this specification and the appended claims, the singular forms "a / an," "another," and "said / the" include plural forms of the referent unless the context clearly dictates otherwise. Unless expressly stated otherwise, the terms "include / comprise / have," "for example," and the like are intended to convey inclusiveness rather than limitation.

[0110] The term "immunogenicity" refers to the ability of a substance, eg a protein or peptide, to provoke an immune response, i.e. the ability to induce antibody production, in particular a humoral or cellular response.

[0111] The term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibodies can be polyclonal mixtures or monoclonal. Antibodies can be intact immunoglobulins of natural or recombinant origin, or immunoreactive portions of intact immunoglobulins. Antibodies can exist in a variety of forms, including, for example, Fv, Fab', F(ab')2, and as single chains.

[0112] The term "antigenicity" refers to the ability of a substance, such as a protein or peptide, to elicit the production of antibodies that specifically bind to it.

[0113] The term "epitope" includes any cluster of protein determinants that specifically bind to an antibody or T-cell receptor. Epitopic determinants generally consist of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains, or combinations thereof) and generally have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0114] The terms "subtype" and "type" are used interchangeably herein to refer to genetic variants of the virus that allow it to be recognized by the immune system as distinct antigens. For example, HPV16 is immunologically distinguishable from HPV33.

[0115] The terms "HPV L1 protein," "HPV," and "human papillomavirus," as used herein, refer to a non-enveloped, double-stranded DNA virus of the papillomavirus family. Its genome is circular and approximately 8 kilobase pairs in size. Most HPVs encode eight major proteins: six in the "early" region (E1-E2) and two in the "late" region (L1 (major capsid protein) and L2 (minor capsid protein)). Over 120 HPV types have been identified, which are identified by numbers (e.g., HPV-16, HPV-18, etc.).

[0116] The term "HPV" or "HPV virus" refers to papillomaviruses of the Papillomaviridae family, which are classified into three regions: (i) the early region (E) (containing six open reading frames E1, E2, E4 to E7, as well as open reading frames E3 and E8, encoding nonstructural proteins associated with viral replication, transcription, and transformation); (ii) the following region (L) (containing reading frames encoding the major capsid protein L1 and the minor capsid protein L2); and (iii) the long control region (LCR), which does not encode any proteins but contains a replication origin and multiple transcription factor binding sites; It is a non-enveloped DNA virus with a double-stranded closed circular DNA genome approximately 8 kb in size, usually classified as

[0117] The terms "HPV L1 protein" and "HPV L2 protein" refer to proteins encoded by the late region (L) of the HPV gene and synthesized late in the HPV infectious cycle. The L2 protein is a minor capsid protein. 72 L1 pentamers form the outer shell of the icosahedral HPV particle, which encloses a closed circular double-stranded DNA minichromosome.

[0118] The term "virus-like particle" refers to a hollow particle that does not contain viral nucleic acid but contains one or more structural proteins of a virus.

[0119] "HPV pseudoviruses" are an ideal model for neutralizing HPV in vitro, taking advantage of the nonspecific nucleic acid encapsulation properties of HPV VLPs; HPV pseudoviruses are formed by wrapping free DNA or by introducing exogenous plasmids into VLPs composed of intracellularly expressed HPV L1 and L2.

[0120] The "pseudovirus neutralization assay" is a method for evaluating the neutralizing activity of antibodies. After incubating the serum of immunized animals with a predetermined amount of pseudovirus to infect cells, as serum neutralizing antibodies increase, the amount of cells decreases, which shows a linear negative correlation within a certain range. Therefore, the neutralizing activity of antibodies in the serum can be evaluated by measuring the change in the amount of cells.

[0121] The terms "fragment thereof" or "variant thereof" refer to deletions, insertions, and / or substitutions of the nucleotide or amino acid sequences of the present invention. Preferably, fragments or variants of the polypeptides provided by the present invention have the ability to elicit a humoral and / or cellular immune response in animals or humans.

[0122] The term "chimeric" means that polypeptide or nucleotide sequences derived from different parent molecules are joined together by -CO-NH- or 3',5'-phosphodiester bonds, respectively. Preferably, such sequences are immediately adjacent to each other without being spaced apart by additional linker sequences.

[0123] The term "truncation" refers to the removal of one or more amino acids from the N-terminus and / or C-terminus of a polypeptide, or the internal deletion of one or more amino acids from a polypeptide.

[0124] The term "nuclear localization sequence" refers to an amino acid sequence that directs a protein into the nucleus. In some HPV L1 proteins, two tight clusters of basic residues (i.e., nuclear localization sequences) (e.g., one is KRKR, KRKK, KRKRK, KRKKRK, KRVKRRK, etc., and the other is KR, RKR, KRK, etc.) have a spacer region of 10 to 14 amino acids between them. The clusters of basic residues belong to the nuclear localization sequence. In some other HPV L1 proteins, the nuclear localization sequence is a tight cluster of basic residues formed by arginine and / or lysine. Examples of nuclear localization sequences include, but are not limited to, the clusters of basic residues described above. See Jun Yang et al., Predicting the Nuclear Localization Signals of 107 Types of HPV L1 Proteins by Bioinformatic Analysis, Genomics, Proteomics & Bioinformatics Volume 4, Issue 1, 2006, pp. 34-41, the entire contents of which are incorporated herein by reference.

[0125] The term "functional variant" refers to a version of a polypeptide or protein that retains a desired activity or property after truncation, mutation, deletion, and / or addition.

[0126] "Sequence identity" between two polypeptide or nucleic acid sequences is expressed as the number of identical residues between the sequences as a percentage of the total number of residues and is calculated based on the size of the shorter of the molecules being compared. When calculating percent identity, the compared sequences are matched to maximize the match between the sequences, filling in gaps (if any) in the match using a specific algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-410). These programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources. The well-known Smith-Waterman algorithm can also be used to determine identity.

[0127] Non-essential amino acids may be conservatively substituted without affecting the normal function of the protein. Conservative substitution means replacing an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables presenting similar amino acids are well known in the art. For example, in some embodiments, the amino acid groups presented in Tables 1-3 are considered to be conservative substitutions for each other.

[0128] [Table 1]

[0129] [Table 2]

[0130] [Table 3]

[0131] The term "amino acid" refers to the 20 common naturally occurring amino acids, including alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0132] As used herein, a "physiologically acceptable carrier" is non-toxic to cells or mammals at the dosages and concentrations used. It is usually a pH-buffered aqueous solution, non-limiting examples of which include buffers, antioxidants, oligopeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium, and / or non-ionic surfactants.

[0133] The term "adjuvant" refers to a compound or mixture that enhances the immune response. In particular, vaccines may contain adjuvants. Adjuvants used in the present invention may include, but are not limited to, one or more of the following: mineral-containing adjuvant compositions, oil emulsion adjuvants, saponin adjuvant formulations, and bacterial or pathogenic derivatives.

[0134] The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that autonomously replicate nucleic acid constructs and vectors that integrate into the genome of a host cell into which the vector is introduced. Certain vectors are capable of inducing expression of nucleic acids to which they are operatively linked.

[0135] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, as well as the progeny of such a cell. Host cells include "transformants" (or "transformed cells"), "transfectants" (or "transfected cells"), or "infectants" (or "infected cells"), each of which includes the primary transformed, transfected, or infected cell, and progeny derived therefrom. Such progeny may not be identical in nucleic acid content to the parent cell and may contain mutations.

[0136] The amount administered is preferably a "prophylactically effective amount" (as used herein, prophylaxis can be considered as treatment, and the two can be used interchangeably), sufficient to show benefit to an individual. [Example]

[0137] Example 1 Construction of chimeric genes Example 1.1 Construction of a chimeric gene in which the C-terminus of HPV6 L1 is replaced with the C-terminus of HPV33 L1 1.1.1 Construction of pFB-HPV6 L1 as a template The HPV6 L1 gene, containing KpnI and XbaI cleavage sites at both ends of the synthesized sequence, was synthesized by Thermo Fisher Scientific (formerly Invitrogen (Shanghai) Trading Co.). The sequence is shown in SEQ ID NO: 5. Plasmid pcDNA3-HPV6-L1, containing the nucleotide sequence encoding amino acids 1 to 500 of HPV6 L1, was obtained by ligating the synthesized gene fragment with the pcDNA3 vector (supplier: Thermo Fisher Scientific) at the KpnI and XbaI cleavage sites.

[0138] The resulting pcDNA3-HPV6-L1 plasmid was subjected to double enzyme digestion with KpnI and XbaI to obtain the HPV6 L1 (1-500) gene fragment, which was then ligated into the KpnI / XbaI double-digested pFastBac™1 vector (supplier: Thermo Fisher Scientific) to obtain a rod vector containing the HPV6 L1 (1-500) gene fragment, designated pFB-HPV6 L1.

[0139] 1.1.2 Construction of pFB-HPV33 L1 as a template The HPV33 L1 gene, containing KpnI and XbaI cleavage sites at both ends of the synthesized sequence, was synthesized by Thermo Fisher (formerly Invitrogen (Shanghai) Trading Co.). The sequence is shown as SEQ ID NO: 6. Plasmid pcDNA3-HPV33-L1, containing the nucleotide sequence encoding amino acids 1 to 499 of HPV33 L1, was obtained by ligating the synthesized gene fragment with the pcDNA3 vector (supplier: Thermo Fisher) at the KpnI and XbaI cleavage sites.

[0140] The pcDNA3-HPV33-L1 plasmid was subjected to double enzyme digestion with KpnI and XbaI to obtain a fragment of the HPV33 L1 (1-499) gene, which was then ligated into a KpnI and XbaI double-digested pFastBac™1 vector (supplier: Thermo Fisher Scientific) to obtain a rod vector containing the HPV33 L1 (1-499) gene fragment, designated pFB-HPV33 L1.

[0141] 1.1.3 Construction of pFB-HPV6 L1:33C The chimeric gene in which the HPV6 L1 C-terminus was replaced with the HPV33 L1 C-terminus, i.e., the constructed recombinant plasmid pFB-HPV6 L1, was used as a gene template to amplify a 1426 bp gene fragment using primers F1 and R1. Primer sequence F1 is shown in SEQ ID NO:7, and R1 is shown in SEQ ID NO:8.

[0142] This gene fragment contains a fragment encoding amino acids 1 to 469 of HPV6 L1, which overlaps by 10 bases with the gene fragment encoding amino acids 474 to 499 of HPV33 L1, and a fragment (GGTAC^C) at the KpnI digestion site. The amplified sequence is shown in SEQ ID NO:9.

[0143] PCR amplification parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 69°C for 15 s, 30 cycles at 1 kb / min at 72°C; extension at 72°C for 5 min; termination at 16°C.

[0144] The recombinant plasmid pFB-HPV33 L1 was used as a gene template to amplify a 101 bp gene fragment using primers F2 and R2. The sequence of the F2 primer is shown in SEQ ID NO: 10, and the primer sequence of R2 is shown in SEQ ID NO: 11.

[0145] This gene fragment contains a gene fragment encoding the C-terminal 26 amino acids (474-499) of HPV33 L1, which overlaps by 10 bp with a gene fragment encoding the C-terminal amino acids 1-469 of HPV6 L1, and an XbaI (T^CTAGA) digestion site. The amplified sequence is shown in SEQ ID NO: 12.

[0146] PCR amplification parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 69°C for 15 s, 30 cycles at 1 kb / min at 72°C; extension at 72°C for 5 min; termination at 16°C.

[0147] PCR ligated sequence: The ligation primers were F1 and R2, and the fragments amplified using the above primers (amplified fragments of F1 and R1, amplified fragments of F2 and R2) were used as templates.

[0148] PCR ligation parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 52°C for 15 s, 5 cycles at 72°C at 1 kb / min; denaturation at 98°C for 10 s, annealing at 68°C for 15 s, 1 kb / min at 72°C for 25 cycles; extension at 72°C for 5 min; termination at 16°C.

[0149] The final result was a nucleotide sequence, SEQ ID NO: 4, encoding amino acids 1 to 469 of HPV6 L1 and the C-terminal 26 amino acids (aa 474 to 499) of HPV33 L1, with KpnI and XbaI cleavage sites at both ends (hereafter referred to as the ligated sequence).

[0150] The recombinant plasmid pFB-HPV6 L1:33C was obtained by double digesting the pFastBac™1 vector and the ligated sequence fragment with KpnI+XbaI enzymes to obtain pFB-HPV6 L1:33C (a chimeric gene in which the C-terminus of HPV6 L1 was replaced by the C-terminus of HPV33 L1), and cloning the ligated sequence into the pFastBac™1 vector.

[0151] Example 1.2 Construction of a chimeric gene in which the C-terminus of HPV11 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 2 for relevant sequences.

[0152] Example 1.3 Construction of a chimeric gene in which the C-terminus of HPV16 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 3 for relevant sequences.

[0153] Example 1.4 Construction of a chimeric gene in which the C-terminus of HPV18 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 4 for relevant sequences.

[0154] Example 1.5 Construction of a chimeric gene in which the C-terminus of HPV31 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 5 for relevant sequences.

[0155] Example 1.6 Construction of the HPV33 L1 gene 1.6.1 Preparation of PFB-HPV33L1 gene The HPV33 L1 gene, containing KpnI and XbaI cleavage sites at both ends of the synthesized sequence, was synthesized by Thermo Fisher Scientific (formerly Invitrogen (Shanghai) Trading Co.). The sequence is shown in SEQ ID NO: 68. Plasmid pcDNA3-HPV33-L1, containing the nucleotide sequence encoding amino acids 1 to 499 of HPV33 L1, was obtained by ligating the synthesized gene fragment with the pcDNA3 vector (supplier: Thermo Fisher Scientific) at the KpnI and XbaI cleavage sites.

[0156] The resulting pcDNA3-HPV33-L1 plasmid was subjected to double enzyme digestion with KpnI and XbaI to obtain the HPV33 L1 gene fragment (1-499). This fragment was then ligated with the KpnI / XbaI double-digested pFastBac™1 vector (supplier: Thermo Fisher Scientific) to obtain a rod vector containing the HPV33 L1 (1-499) gene fragment, designated pFB-HPV33 L1.

[0157] Example 1.7 Construction of a chimeric gene in which the C-terminus of HPV35 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 7 for relevant sequences.

[0158] Example 1.8 Construction of a chimeric gene in which the C-terminus of HPV39 L1 is replaced by the C-terminus of HPV59 L1 1.8.1 Construction of pFB-HPV39 L1 used as template The HPV39 L1 gene, containing KpnI and XbaI cleavage sites at both ends of the synthesized sequence, was synthesized by Thermo Fisher (formerly Invitrogen (Shanghai) Trading Co.). The sequence is shown as SEQ ID NO: 86. Plasmid pcDNA3-HPV39-L1, containing the nucleotide sequence encoding amino acids 1 to 505 of HPV39 L1, was obtained by ligating the synthesized gene fragment with the pcDNA3 vector (supplier: Thermo Fisher) at the KpnI and XbaI cleavage sites.

[0159] The pcDNA3-HPV39-L1 plasmid was double-digested with KpnI and XbaI to obtain a fragment (1-505) of the HPV39 L1 gene, which was then ligated into a KpnI- and XbaI-digested pFastBac™1 vector (available from Thermo Fisher Scientific) to obtain a rod vector containing the HPV39 L1 (1-505) gene fragment, designated pFB-HPV39 L1.

[0160] 1.8.2 Construction of pFB-HPV59 L1 as a template The HPV59 L1 gene, containing KpnI and XbaI cleavage sites at both ends of the synthesized sequence, was synthesized by Thermo Fisher (formerly Invitrogen (Shanghai) Trading Co.). The sequence is shown as SEQ ID NO: 87. Plasmid pcDNA3-HPV59-L1, containing the nucleotide sequence encoding amino acids 1 to 508 of HPV59 L1, was obtained by ligating the synthesized gene fragment with the pcDNA3 vector (supplier: Thermo Fisher) at the KpnI and XbaI cleavage sites.

[0161] The pcDNA3-HPV59-L1 plasmid was double-digested with KpnI and XbaI to obtain a fragment of the HPV59 L1 (1-508) gene, which was then ligated into a KpnI- and XbaI-digested pFastBac™1 vector (available from Thermo Fisher Scientific) to obtain a rod vector containing the HPV59 L1 (1-508) gene fragment, designated pFB-HPV59 L1.

[0162] 1.8.3 Construction of pFB-HPV39 L1:59C Chimeric gene in which the C-terminus of HPV39 L1 was replaced with the C-terminus of HPV59 L1: The constructed recombinant plasmid pFB-HPV39 L1 was used as a gene template to amplify a 1428 bp gene fragment using primers F1 and R1. The primer sequence F1 is shown in SEQ ID NO:88, and the primer sequence R1 is shown in SEQ ID NO:89.

[0163] This fragment contains a fragment encoding amino acids 1 to 469 of HPV39 L1, which overlaps by 12 bases with a fragment encoding amino acids 471 to 508 of HPV59 L1, and a segment of the KpnI digestion site (GGTAC^C). The amplified sequence is shown in SEQ ID NO:90.

[0164] PCR amplification parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 69°C for 15 s, 30 cycles at 1 kb / min at 72°C; extension at 72°C for 5 min; termination at 16°C.

[0165] The recombinant plasmid pFB-HPV59 L1 was used as a gene template to amplify a 139 bp long gene fragment using primers F2 and R2. Primer sequences F2 is shown in SEQ ID NO:91 and R2 is shown in SEQ ID NO:92.

[0166] This gene fragment contains a gene fragment encoding the 38 C-terminal amino acids of HPV59 L1 (aa 471 to 508) that overlaps by 12 bp with the gene fragment encoding amino acids 1 to 469 of HPV39 L1, as well as an XbaI (T^CTAGA) digestion site. The amplified sequence is shown in SEQ ID NO: 93.

[0167] PCR amplification parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 69°C for 15 s, 30 cycles at 1 kb / min at 72°C; extension at 72°C for 5 min; termination at 16°C.

[0168] PCR ligated sequences The ligation primers were F1 and R2, and the fragments amplified by using the above primers (F1 and R1 amplified fragments, F2 and R2 amplified fragments) were used as templates.

[0169] PCR ligation parameters: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 52°C for 15 s, 5 cycles at 72°C at 1 kb / min; denaturation at 98°C for 10 s, annealing at 68°C for 15 s, 1 kb / min at 72°C for 25 cycles; extension at 72°C for 5 min; termination at 16°C.

[0170] The final result was a nucleotide sequence, SEQ ID NO: 85, encoding amino acids 1 to 469 of HPV39 L1 and the C-terminal 38 amino acids (471 to 508) of HPV59 L1 with KpnI and XbaI enzyme cleavage sites at both ends (hereafter referred to as the ligated sequence).

[0171] To obtain pFB-HPV39 L1:59C, a chimeric gene in which the C-terminus of HPV39 L1 was replaced by the C-terminus of HPV59 L1, the recombinant plasmid pFB-HPV39 L1:59C was obtained by double-digesting the pFastBac™1 vector and the ligated sequence fragment with KpnI+XbaI enzymes and cloning the ligated sequence into the pFastBac™1 vector.

[0172] Example 1.9 Construction of a chimeric gene in which the C-terminus of HPV45 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 9 for relevant sequences.

[0173] Example 1.10 Construction of a chimeric gene in which the C-terminus of HPV51 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 10 for relevant sequences.

[0174] Example 1.11 Construction of a chimeric gene in which the C-terminus of HPV52 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 11 for relevant sequences.

[0175] Example 1.12 Construction of a chimeric gene in which the C-terminus of HPV56 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 12 for relevant sequences.

[0176] Example 1.13 Construction of a chimeric gene in which the C-terminus of HPV58 L1 is replaced by the C-terminus of HPV33 L1 The experimental methods and procedures were the same as in Example 1.1. See Appendix 13 for relevant sequences.

[0177] Example 1.14 Construction of the HPV59 L1 gene

[0178] Example 1.14.1 Preparation of pFB-HPV59 L1 gene The HPV59 L1 gene containing KpnI and XbaI cleavage sites at both ends of the synthesized sequence was synthesized by Thermo Fisher Scientific (formerly Invitrogen (Shanghai) Trading Co.). The sequence is shown in SEQ ID NO: 162. The synthesized gene fragment was ligated with the pcDNA3 vector (supplier: Thermo Fisher Scientific) at the KpnI and XbaI cleavage sites to obtain the plasmid pcDNA3-HPV59-L1 containing the nucleotide sequence encoding amino acids 1 to 508 of HPV59 L1.

[0179] The resulting pcDNA3-HPV59-L1 plasmid was subjected to double enzyme digestion with KpnI and XbaI to obtain the HPV59 L1 (1-508) gene fragment, which was then ligated into the KpnI / XbaI double-digested pFastBac™1 vector (supplier: Thermo Fisher Scientific) to obtain a rod vector containing the HPV59 L1 (1-508) gene fragment, designated pFB-HPV59 L1.

[0180] Example 2 Recombinant baculovirus packaging Example 2.1 HPV6 L1:33C recombinant baculovirus packaging The recombinant plasmid pFB-HPV6 L1:33C constructed in Example 1 was identified and sequenced to be correct, and transformed into DH10Bac bacterial competent cells (Bac-to-Bac® Kit, purchased from Thermo Fisher Scientific), incubated at 37°C for growth, and incubated in flat-bottom dishes for streaking. White colonies were selected and incubated overnight. The bacterial culture was harvested, and recombinant baculovirus DNA was extracted using the alkaline lysis method.

[0181] The recombinant baculovirus DNA was transfected into insect cells SF9 using a cationic transfection reagent (purchased from Sino Biological Co., Ltd.) to package the recombinant baculovirus virulent strain. The procedure was as follows:

[0182] a. Log-phase SF9 cells were cultured at 0.6 × 10 cells 6 The dishes were inoculated with SF9 cells at a density of 100 cells / dish. The dishes inoculated with SF9 cells were left at room temperature for 2 hours to allow the cells to adhere to the wall of the dish.

[0183] b. The extracted plasmid Bacmid DNA (20 μL) was added to Grace's medium (serum-free, additive-free, purchased from Gibico) (200 μL) and mixed by inverting five times.

[0184] c. 0.2×TF1 (transfection reagent, purchased from Sino Biological Co., Ltd.) (25 μL) was added dropwise to Grace's medium (200 μL) and mixed gently.

[0185] d. Mix b and c. Incubate at room temperature for 15-45 minutes.

[0186] e. During the incubation of DNA with Cellfectin (purchased from Sino Biological Co., Ltd.), the cell supernatant was discarded, and Grace's medium (serum-free and additive-free) (0.8 mL) was added to the dish.

[0187] f. The incubated DNA mixture and the transfection reagent from d were added dropwise to the dish.

[0188] g. Incubated at 27°C for 2 hours.

[0189] h. Discard the cell culture medium and add 2.5 mL / dish of complete growth medium (SCD6 SF + 10% FBS) (SCD6 SF purchased from Sino Biological Company, FBS purchased from Gibico Company).

[0190] i. The culture was carried out at 27°C for 7 days, and then it was observed whether virus infection occurred or not.

[0191] Viral supernatant was collected after visible lesions were observed on the transfected cells, typically after 7–11 days of culture. Viral supernatant, i.e., the P1 generation virus strain of HPV6 L1:33C, was collected aseptically using a pipette. 2 × 10 cells 6 SF9 cells at a density of 1000 / mL were infected with HPV6 L1:33C P1 generation virus strain at a ratio of 1:50 (V / V), cultured at 27°C for 3 days, and centrifuged at 1000g ± 200g for 10 minutes at room temperature. The collected viral supernatant was P2 generation virus, which can be used to infect and purify host cells.

[0192] Example 2.2 Packaging of HPV11 L1:33C recombinant baculovirus The experimental methods and procedures were the same as in Example 2.1.

[0193] Example 2.3 Packaging of HPV16L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0194] Example 2.4 Packaging of HPV18 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0195] Example 2.5 Packaging of HPV31 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0196] Example 2.6 Packaging of HPV33 L1 recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0197] Example 2.7 Packaging of HPV35 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0198] Example 2.8 Packaging of HPV39 L1:59C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0199] Example 2.9 Packaging of HPV45 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0200] Example 2.10 Packaging of HPV51 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0201] Example 2.11 Packaging of HPV52 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0202] Example 2.12 Packaging of HPV56 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0203] Example 2.13 Packaging of HPV58 L1:33C recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0204] Example 2.14 Packaging of HPV59 L1 recombinant baculovirus The experimental methods and procedures were the same as those in Example 2.1.

[0205] Example 3 Expression of chimeric proteins or proteins Example 3.1 HPV6 L1:33C expression High Five cells were infected with the baculovirus containing the HPV6 L1:33C recombinant gene obtained in Example 2 at a ratio of 1:200 (V / V), and the cell pellet was collected by centrifugation at room temperature at 1000 g ± 100 g. The cells were disrupted by sonication at low temperature for 3 minutes, centrifuged at >10,000 g for 10 minutes, and the supernatant was collected for SDS-PAGE. Lane 1: Marker (the marker is a mixture of seven purified proteins with molecular weights ranging from 14.4 kDa to 116 kDa, manufactured by Thermo Scientific); Lane 2: Cell lysate; Lane 3: Lysate supernatant collected by centrifugation.

[0206] The results are shown in Figure 1 A. The HPV6 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0207] Example 3.2 Expression and production of HPV11 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0208] The results are shown in Figure 1B. The HPV11 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0209] Example 3.3 Expression and production of HPV16L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0210] The results are shown in Figure 1 C. The HPV16 L1:33C L1 protein prepared by this method had a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0211] Example 3.4 Expression and production of HPV18 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0212] The results are shown in Figure ID. The HPV18 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0213] Example 3.5 Expression and production of HPV31 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0214] The results are shown in Figure 1E. The HPV31 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0215] Example 3.6 HPV33 L1 expression and production The experimental methods and procedures were the same as in Example 3.1.

[0216] The results are shown in Figure 1 F. The HPV33 L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0217] Example 3.7 Expression and production of HPV35 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0218] The results are shown in Figure 1G. The HPV35 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0219] Example 3.8 Expression and production of HPV39 L1:59C The experimental methods and procedures were the same as in Example 3.1.

[0220] The results are shown in Figure 1H. The HPV39 L1:59C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0221] Example 3.9 Expression and production of HPV45 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0222] The results are shown in Figure 11. The HPV45 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0223] Example 3.10 Expression and production of HPV51 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0224] The results are shown in Figure 1J. The HPV51 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0225] Example 3.11 Expression and production of HPV52 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0226] The results are shown in Figure 1K. The HPV52 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0227] Example 3.12 Expression and production of HPV56 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0228] The results are shown in Figure 1 L. The HPV56 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0229] Example 3.13 Expression and production of HPV58 L1:33C The experimental methods and procedures were the same as in Example 3.1.

[0230] The results are shown in Figure 1 M. The HPV58 L1:33C L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0231] Example 3.14 HPV59 L1 expression and production The experimental methods and procedures were the same as in Example 3.1.

[0232] The results are shown in Figure 1 N. The HPV59 L1 protein prepared by this method has a yield of >100 mg / L and a protein size of approximately 56 KD, making it suitable for mass production.

[0233] Example 4 Preparation of purified virus-like particles Example 4.1 Preparation of purified HPV6 L1:33C virus-like particles HPV6 L1:33C virus-like particles were purified by a two-step chromatography method, namely, HS-MMA method, to purify the supernatant collected in Example 3, and finally obtain highly pure virus-like particles.

[0234] First step chromatography: Media: POROS® 50 HS strong cation exchange media manufactured by Thermo Fisher was used.

[0235] Media volume: Media volume 150mL, linear flow rate 30mL / min.

[0236] Chromatographic conditions: equilibration buffer (pH 6.2, salt concentration is 50 mM phosphate / 0.5 M NaCl); washing buffer (salt concentration is 50 mM phosphate / 0.75 M NaCl, pH 6.2).

[0237] The chromatography column was first equilibrated with 5 CV of equilibration buffer, and then the sample was loaded. After loading, the column was then eluted with 5 CV of equilibration buffer and wash buffer, respectively, to remove protein impurities.

[0238] Elution conditions: 50 mM phosphate buffer, pH 6.2, containing 50 mM arginine hydrochloride, with an elution salt concentration of 1.25 M NaCl was used.

[0239] Second step chromatography Media: MMA ion exchange media manufactured by Bestchrom (Shanghai) Biosciences Co., Ltd. was used.

[0240] Medium volume: The medium volume is 150 mL, while the linear flow rate is 30 mL / min.

[0241] Chromatography conditions: Equilibration buffer: 50 mM PB / 1.25 M NaCl, pH 6.2. The chromatography column was first equilibrated with 4 CV of equilibration buffer, and then the sample was loaded. After loading, protein impurities were washed away with 5 CV of equilibration buffer, and then the target protein was eluted with elution buffer and collected.

[0242] Elution conditions: 100 mM NaAC / 150 mM NaCl / 0.01% Tween 80, pH 4.5.

[0243] Example 4.2 Preparation of purified HPV11 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0244] Example 4.3 Purification and preparation of HPV16L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0245] Example 4.4 Purification and preparation of HPV18 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0246] Example 4.5 Purification and preparation of HPV31 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0247] Example 4.6 Preparation of purified HPV33 L1 virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0248] Example 4.7 Preparation of purified HPV35 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0249] Example 4.8 Preparation of purified HPV39 L1:59C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0250] Example 4.9 Preparation of purified HPV45 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0251] Example 4.10 Preparation of purified HPV51 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0252] Example 4.11 Preparation of purified HPV52 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0253] Example 4.12 Preparation of purified HPV56 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0254] Example 4.13 Preparation of purified HPV58 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0255] Example 4.14 Preparation of purified HPV59 L1 virus-like particles The experimental methods and procedures were the same as in Example 4.1.

[0256] Example 5 Morphological observation of virus-like particles Example 5.1 Morphological observation of HPV6 L1:33C virus-like particles A sample (10 μL) was taken for transmission electron microscopy. The sample was fixed on a carbon-coated copper grid for 2 minutes, the remaining liquid was absorbed with filter paper, and then stained twice with phosphotungstic acid (Beijing Electron Microscopy China Technology Co., Ltd., 2% concentration, pH 6.5) for 30 seconds each time. The remaining staining solution was absorbed with filter paper, and the sample was allowed to dry before being observed by transmission electron microscopy. The transmission electron microscope (Brand: Hitachi, Model Number: H-7650) was operated at 80 KV and 80,000x magnification.

[0257] Electron microscopy observations are shown in Figure 2 A. As can be seen in Figure 2 A, the C-terminally modified HPV6 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0258] Example 5.2 Morphological observation of HPV11 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0259] Electron microscopy observations are shown in Figure 2B. As can be seen in Figure 2B, the C-terminally modified HPV11 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0260] Example 5.3 Morphological observation of HPV16L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0261] Electron microscopy observations are shown in Figure 2C. As can be seen in Figure 2C, the C-terminally modified HPV16L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0262] Example 5.4 Morphological observation of HPV18 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0263] Electron microscopy observations are shown in Figure 2D. As can be seen in Figure 2D, the C-terminally modified HPV18 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0264] Example 5.5 Morphological observation of HPV31 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0265] Electron microscopy observations are shown in Figure 2E. As can be seen in Figure 2E, the C-terminally modified HPV31 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0266] Example 5.6 Morphological observation of HPV33 L1 virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0267] Electron microscopy observations are shown in Figure 2F. As can be seen in Figure 2F, the C-terminally modified HPV33 L1 can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0268] Example 5.7 Morphological observation of HPV35 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0269] Electron microscopy observations are shown in Figure 2G. As can be seen in Figure 2G, the C-terminally modified HPV35 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0270] Example 5.8 Morphological observation of HPV39 L1:59C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0271] Electron microscopy observations are shown in Figure 2H. As can be seen in Figure 2H, the C-terminally modified HPV39 L1:59C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0272] Example 5.9 Morphological observation of HPV45 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0273] Electron microscopy observations are shown in Figure 2 I. As can be seen in Figure 2 I, the C-terminally modified HPV45 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0274] Example 5.10 Morphological observation of HPV51 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0275] Electron microscopy observations are shown in Figure 2J. As can be seen in Figure 2J, the C-terminally modified HPV51 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0276] Example 5.11 Morphological observation of HPV52 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0277] Electron microscopy observations are shown in Figure 2K. As can be seen in Figure 2K, the C-terminally modified HPV52 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0278] Example 5.12 Morphological observation of HPV56 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0279] Electron microscopy observations are shown in Figure 2 L. As can be seen in Figure 2 L, the C-terminally modified HPV56 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0280] Example 5.13 Morphological observation of HPV58 L1:33C virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0281] Electron microscopy observations are shown in Figure 2M. As can be seen in Figure 2M, the C-terminally modified HPV58 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0282] Example 5.14 Morphological observation of HPV59 L1 virus-like particles The experimental methods and procedures were the same as in Example 5.1.

[0283] Electron microscopy observations are shown in Figure 2N. As can be seen in Figure 2N, the C-terminally modified HPV59 L1:33C can form uniformly sized virus-like particles with an average diameter of approximately 60 nm.

[0284] Example 6 Immunogenicity evaluation of virus-like particles in animals Example 6.1 Immunogenicity evaluation of HPV6 L1:33C virus-like particles in animals 6.1.1 Modeling pseudovirus-neutralizing cells HPV is difficult to culture in vitro and has strong host specificity, making it difficult to replicate in organisms other than humans, and therefore there are no suitable animal models. Therefore, there is a need to establish a suitable and effective in vitro neutralization experimental model for evaluating the immunoprotective properties of vaccines.

[0285] HPV pseudoviruses are ideal models for in vitro neutralization of HPV: due to the non-specific nucleic acid encapsulation property of HPV VLPs, HPV pseudoviruses can be formed from VLPs (composed of intracellularly expressed HPV L1 and L2) by encapsulating free DNA or by introducing exogenous plasmids.

[0286] The immunogenicity of immunized animal serum samples was analyzed by pseudovirus neutralization assay.Animals immunized with HPV6 virus-like particles can produce neutralizing antibodies against HPV6, and these neutralizing antibodies can neutralize HPV6 pseudovirus.When immunized animal serum is incubated with a predetermined amount of pseudovirus and then infected with cells, the increase in neutralizing antibodies in serum leads to a decrease in the number of cells capable of expressing GFP fluorescence, which shows a linear negative correlation within a certain range.Therefore, the neutralizing activity of antibodies in serum can be evaluated by detecting changes in the number of cells expressing GFP.

[0287] Pseudovirus construction method: The HPV6 pCMV3-3-HPV6 L1+L2 (L1 sequence derived from Uniprot P69898, L2 sequence derived from Uniprot Q84297) plasmid (purchased from Sino Biological) and a fluorescent plasmid (PSEU-GFP Spark, purchased from Sino Biological) were co-transfected into 293FT adherent cells (purchased from Thermo Fisher Scientific). For specific methods, please refer to the published literature (Pastrana DV, Buck CB, Pang YS, Thompson CD, Castle PE, FitzGerald PC, Kjaer SK, Lowy DR, Schiller J T. Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18. [J] Virology 2004, 321:205-216). The pseudovirus supernatant was collected, aliquoted, and stored in a -80°C freezer for stock.

[0288] 6.1.2 Immunoprotective Evaluation of HPV6 L1:33C Virus-Like Particles in Animals

[0289] Immunization procedure in mice:

[0290] HPV6 L1:33C virus-like particles were absorbed onto aluminum phosphate adjuvant, mixed, and used to immunize mice (10 mice total) at a dose of 0.15 μg / 200 μL per mouse. Mice were immunized with the diluted samples on days 0, 7, and 21, while control mice were immunized with blank serum. Blood was collected from the eyes of the mice on day 28, and serum was isolated for pseudovirus neutralization titer assays.

[0291] Mouse EC50 assay: Mouse serum was inactivated at 56°C for 30 minutes, centrifuged at 6000g for 5 minutes, and the supernatant was collected for assay. For 4 to 8 hours prior to the assay, 293FT cells were inoculated into 96-well plates at a density of 15,000 cells / well and incubated at 37°C in a CO2 incubator containing 5% CO2. Postimmunization mouse serum and blank control serum were serially diluted with neutralizing medium and then mixed with the HPV6 pseudovirus prepared in Section 6.1 in a 1:1 volumetric ratio and incubated at 2 to 8°C for 1 hour. 100 μL of the mixture was then added per well to 293FT cells (previously inoculated for 4 to 8 hours). Each sample was used in duplicate, and a blank serum control group, a pseudovirus-positive control group, and a pseudovirus-negative control group were used. The pseudovirus-infected cells were incubated for 62–96 hours at 37°C in a CO2 incubator containing 5% CO2, photographed by fluorescent scanning, and measured using an ELISPOT analyzer (Model No.: S6 Universal-V Analyzer, Manufacturer: CTL). The half-efficacy dilution (EC50) was calculated for each mouse serum sample based on the neutralization inhibition of each sample, i.e., the highest serum dilution required for 50% neutralization inhibition, according to the Reed-Muench method.

[0292] The results of the HPV6 serum pseudovirus neutralization titer assay are detailed in Table 4.

[0293] [Table 4]

[0294] Notes: 1. Number of animals, N = 10. 2. GMT (Geometric Mean Titer): Geometric mean titer. 3. SEM (Standard Error of Mean): Standard error.

[0295] The above evaluation results indicate that the HPV6 L1:33C virus-like particles prepared according to the present invention have good immunogenicity and can generate high-titer neutralizing antibodies in animals (which can be used to prepare vaccines for preventing HPV infection).

[0296] Example 6.2 Immunogenicity evaluation of HPV11 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P04012 and the L2 sequence was derived from Uniprot P04013.

[0297] The results of the HPV11 serum pseudovirus neutralization titer assay are detailed in Table 5.

[0298] [Table 5]

[0299] The above evaluation results indicate that the HPV11 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0300] Example 6.3 Immunogenicity evaluation of HPV16L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P03101 and the L2 sequence was derived from Uniprot P03107.

[0301] The results of the HPV16 serum pseudovirus neutralization titer assay are detailed in Table 6.

[0302] [Table 6]

[0303] The above evaluation results indicate that the HPV16L1:33C virus-like particles prepared according to the present invention have good immunogenicity and can generate high titers of neutralizing antibodies in animals (which can be used to prepare vaccines for preventing HPV infection).

[0304] Example 6.4 Immunogenicity evaluation of HPV18 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot Q80B70 and the L2 sequence was derived from Uniprot P06793.

[0305] The results of the HPV18 serum pseudovirus neutralization titer assay are detailed in Table 7.

[0306] [Table 7]

[0307] The above evaluation results indicate that the HPV18 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0308] Example 6.5 Immunogenicity evaluation of HPV31 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P17388 and the L2 sequence was derived from Uniprot P17389.

[0309] The results of the HPV31 serum pseudovirus neutralization titer assay are detailed in Table 8.

[0310] [Table 8]

[0311] The above evaluation results indicate that the HPV31 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0312] Example 6.6 Immunogenicity evaluation of HPV33 L1 virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P06416 and the L2 sequence was derived from Uniprot P06418.

[0313] The results of the HPV33 serum pseudovirus neutralization titer assay are detailed in Table 9.

[0314] [Table 9]

[0315] The above evaluation results indicate that the HPV33 L1 virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0316] Example 6.7 Immunogenicity evaluation of HPV35 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P27232 and the L2 sequence was derived from Uniprot P27234.

[0317] The results of the HPV35 serum pseudovirus neutralization titer assay are detailed in Table 10.

[0318] [Table 10]

[0319] The above evaluation results indicate that the HPV35 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0320] Example 6.8 Immunogenicity evaluation of HPV39 L1:59C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P24838 and the L2 sequence was derived from Uniprot P24839.

[0321] The results of the HPV39 serum pseudovirus neutralization titer assay are detailed in Table 11.

[0322] [Table 11]

[0323] The above evaluation results indicate that the HPV39 L1:59C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0324] Example 6.9 Immunogenicity evaluation of HPV45 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P36741, and the L2 sequence was derived from Uniprot P36761.

[0325] The results of the HPV45 serum pseudovirus neutralization titer assay are detailed in Table 12.

[0326] [Table 12]

[0327] The above evaluation results indicate that the HPV45 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0328] Example 6.10 Immunogenicity evaluation of HPV51 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P26536 and the L2 sequence was derived from Uniprot P26539.

[0329] The results of the HPV51 serum pseudovirus neutralization titer assay are detailed in Table 13.

[0330] [Table 13]

[0331] The above evaluation results indicate that the HPV51 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0332] Example 6.11 Immunogenicity evaluation of HPV52 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot Q05138 and the L2 sequence was derived from Uniprot F8S4U2.

[0333] The results of the HPV52 serum pseudovirus neutralization titer assay are detailed in Table 14.

[0334] [Table 14]

[0335] The above evaluation results indicate that the HPV52 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0336] Example 6.12 Immunogenicity evaluation of HPV56 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P36743 and the L2 sequence was derived from Uniprot P36765.

[0337] The results of the HPV56 serum pseudovirus neutralization titer assay are detailed in Table 15.

[0338] [Table 15]

[0339] The above evaluation results indicate that the HPV56 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0340] Example 6.13 Immunogenicity evaluation of HPV58 L1:33C virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot P26535 and the L2 sequence was derived from Uniprot B6ZB12.

[0341] The results of the HPV58 serum pseudovirus neutralization titer assay are detailed in Table 16.

[0342] [Table 16]

[0343] The above evaluation results indicate that the HPV58 L1:33C virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0344] Example 6.14 Immunogenicity evaluation of HPV59 L1 virus-like particles in animals The experimental methods and procedures were the same as in Example 6.1. The L1 sequence was derived from Uniprot Q81971 and the L2 sequence was derived from Uniprot Q81970.

[0345] The results of the HPV59 serum pseudovirus neutralization titer assay are detailed in Table 17.

[0346] [Table 17]

[0347] The above evaluation results indicate that the HPV59 L1 virus-like particles prepared according to the present invention have good immunogenicity, can generate high titers of neutralizing antibodies in animals, and can be used to prepare vaccines for preventing HPV infection.

[0348] Example 6.15 Immunogenicity evaluation of a 14-valent immunogenic composition consisting of virus-like particles in mice Immunization procedure in mice: A sample composed of the above virus-like particles of 14 types (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) was sterilely diluted with aluminum phosphate adjuvant (e.g., an HPV type accounting for 20 μg in the sample was diluted 80-fold to 0.5 μg / mL; different amounts will result in correspondingly different concentrations of other HPV types in the sample). Each mouse was immunized with 200 μL of the diluted virus-like particle sample, at the doses shown in Table 18. Six- to eight-week-old SPF-grade female Balb / c mice were divided into several groups (experimental and control groups) consisting of 10 mice per group, and immunized with the diluted samples on days 0, 7, and 21, respectively, except for control mice, which were immunized with blank serum. On day 28, blood was collected from the eyes of the mice and serum was isolated for pseudovirus neutralization titer assays.

[0349] [Table 18]

[0350] Serum neutralization titer assay in immunized mice using pseudovirus neutralization assay: Mouse serum was inactivated at 56°C for 30 minutes, centrifuged at 6000g for 5 minutes, and the supernatant was collected for assay. 293FT cells were inoculated into 96-well plates at a density of 15,000 cells / well and incubated at 37°C in a CO2 incubator containing 5% CO2 for 4-8 hours prior to assay. The immunized mouse serum and blank control serum were serially diluted with neutralizing medium and then mixed with diluted HPV type 14 pseudovirus samples (HPV type 14 pseudoviruses are HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 pseudoviruses prepared in Examples 6.1 to 6.14) at a 1:1 volume ratio and incubated at 2 to 8°C for 1 hour. 100 μL / well of the mixture was then added to 293FT cells (previously inoculated for 4 to 8 hours). Each sample was used in duplicate, and a blank serum control group, a pseudovirus-positive control group, and a pseudovirus-negative control group were used. The pseudovirus-infected cells were incubated for 62-96 hours at 37°C in a CO2 incubator containing 5% CO2. Fluorescence scanning photography was performed and the results were analyzed using an ELISPOT analyzer (Model No. S6 Universal-V Analyzer, Manufacturer: CTL). The maximum serum dilution required for 50% neutralization inhibition, i.e., the median effective dilution (EC50), was calculated for each mouse serum sample according to the Reed-Muench method. The results are shown in Figure 3.

[0351] As demonstrated in Figure 3, the 14-valent immunogenic composition of virus-like particles can generate high titers of neutralizing antibodies in animals and can be used to prepare a vaccine to prevent HPV infection.

[0352] Comparative Example 1: Expression of C-terminally truncated HPV16 L1 (aa 1-474) The present inventors attempted to truncate the C-terminus of HPV16 L1 by 31 amino acids and designated it HPV16 L1(1-474) (SEQ ID NO: 27). However, tests showed that the truncated HPV16 L1(1-474) protein, although highly expressed, was highly insoluble and difficult to extract and purify. Detailed results of expression and extraction are shown in Figure 4.

[0353] Although the present invention has been described in detail above by way of examples and embodiments, it is intended to be easy to understand. It is obvious to those skilled in the art that various modifications and improvements can be made to the technical solutions of the present invention without departing from the spirit or scope of the appended claims.

[0354] Appendix 1: Sequence Listing - Chimeric Human Papillomavirus Type 6 L1 Protein

[0355] [Table 19A]

[0356] [Table 19B]

[0357] [Table 19C]

[0358] [Table 19D]

[0359] [Table 19E]

[0360] [Table 19F]

[0361] Appendix 2: Sequence Listing - Chimeric Human Papillomavirus Type 11 L1 Protein

[0362] [Table 20A]

[0363] [Table 20B]

[0364] [Table 20C]

[0365] [Table 20D]

[0366] [Table 20E]

[0367] [Table 20F]

[0368] Appendix 3: Sequence Listing - Chimeric Human Papillomavirus Type 16 L1 Protein

[0369] [Table 21A]

[0370] [Table 21B]

[0371] [Table 21C]

[0372] [Table 21D]

[0373] [Table 21E]

[0374] [Table 21F]

[0375] Appendix 4: Sequence Listing - Chimeric Human Papillomavirus Type 18 L1 Protein

[0376] [Table 22A]

[0377] [Table 22B]

[0378] [Table 22C]

[0379] [Table 22D]

[0380] [Table 22E]

[0381] [Table 22F]

[0382] Appendix 5: Sequence Listing - Chimeric Human Papillomavirus Type 31 L1 Protein

[0383] [Table 23A]

[0384] [Table 23B]

[0385] [Table 23C]

[0386] [Table 23D]

[0387] [Table 23E]

[0388] [Table 23F]

[0389] Appendix 6: Sequence Listing - Human Papillomavirus Type 33 L1 Protein

[0390] [Table 24A]

[0391] [Table 24B]

[0392] [Table 24C]

[0393] Appendix 7: Sequence Listing - Chimeric Human Papillomavirus Type 35 L1 Protein

[0394] [Table 25A]

[0395] [Table 25B]

[0396] [Table 25C]

[0397] [Table 25D]

[0398] [Table 25E]

[0399] [Table 25F]

[0400] Appendix 8: Sequence Listing - Chimeric Human Papillomavirus Type 39 L1 Protein

[0401] [Table 26A]

[0402] [Table 26B]

[0403] [Table 26C]

[0404] [Table 26D]

[0405] [Table 26E]

[0406] [Table 26F]

[0407] Appendix 9: Sequence Listing - Chimeric Human Papillomavirus Type 45 L1 Protein

[0408] [Table 27A]

[0409] [Table 27B]

[0410] [Table 27C]

[0411] [Table 27D]

[0412] [Table 27E]

[0413] [Table 27F]

[0414] Appendix 10: Sequence Listing - Chimeric Human Papillomavirus Type 51 L1 Protein

[0415] [Table 28A]

[0416] [Table 28B]

[0417] [Table 28C]

[0418] [Table 28D]

[0419] [Table 28E]

[0420] [Table 28F]

[0421] Appendix 11: Sequence Listing - Chimeric Human Papillomavirus Type 52 L1 Protein

[0422] [Table 29A]

[0423] [Table 29B]

[0424] [Table 29C]

[0425] [Table 29D]

[0426] [Table 29E]

[0427] [Table 29F]

[0428] Appendix 12: Sequence Listing - Chimeric Human Papillomavirus Type 56 L1 Protein

[0429] [Table 30A]

[0430] [Table 30B]

[0431] [Table 30C]

[0432] [Table 30D]

[0433] [Table 30E]

[0434] [Table 30F]

[0435] Appendix 13: Sequence Listing - Chimeric Human Papillomavirus Type 58 L1 Protein

[0436] [Table 31A]

[0437] [Table 31B]

[0438] [Table 31C]

[0439] [Table 31D]

[0440] [Table 31E]

[0441] [Table 31F]

[0442] Appendix 14: Sequence Listing - Human Papillomavirus Type 59 L1 Protein

[0443] [Table 32A]

[0444] [Table 32B]

[0445] [Table 32C]

Claims

1. A multivalent HPV immunogenic composition for preventing HPV-related diseases or infections, the composition comprising: (1) a chimeric HPV L1 protein consisting of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58; and (2) an HPV type 33 L1 protein and an HPV type 59 L1 protein; The amino acid sequences of the chimeric HPV L1 proteins of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58 are set forth in SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 55, SEQ ID NO: 71, SEQ ID NO: 84, SEQ ID NO: 97, SEQ ID NO: 110, SEQ ID NO: 123, SEQ ID NO: 136, and SEQ ID NO: 149, respectively; A multivalent HPV immunogenic composition, wherein the amino acid sequences of HPV type 33 L1 protein and HPV type 59 L1 protein are set forth in SEQ ID NO: 66 and SEQ ID NO: 160, respectively.

2. 10. The multivalent HPV immunogenic composition of claim 1, further comprising a physiologically acceptable carrier, and optionally an adjuvant.

3. 3. The multivalent HPV immunogenic composition of claim 2, wherein the adjuvant is an aluminum phosphate adjuvant.

4. 10. Use of a multivalent HPV immunogenic composition according to any one of claims 1 to 3 in the preparation of a vaccine or drug for the prevention of an HPV-related disease or infection.

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